The sodium pump has been the target for the therapeutic treatment of congestive heart failure with cardiac glycosides for more than a century. Unfortunately, there is still very little known about the mechanism of cardiac glycoside inhibition. Experimental studies over the past three decades have established some relationships between the biochemical reactions catalyzed by the sodium pump and the transport reactions it mediates. However, the molecular mechanism by which the hydrolysis of ATP is coupled to the "uphill" movement of ions remains a mystery. This probably stems from the fact that there remain key components about the structure-function relationship of this protein yet to be elucidated. For example, why are the Na,K and H,K-ATPases the only members of this protein superfamily to have an obligatory alpha- and beta-subunit? Our long term goal is to gain a complete understanding of the sodium pump transport mechanism, as well as defining the other cellular roles mediated by Na,K-ATPase. For example, the Na pump has been shown to associate with several cytosolic proteins and may serve as a plasma membrane docking site for these signaling molecules (e.g. PI-3 kinase). However, before we can attempt to resolve the basis for these hetero protein-protein interactions, we must first identify the characteristics for Na pump assembly and trafficking. Indeed, there is still considerable debate over the quaternary structure of the Na,K-ATPase, a question directly examined in this proposal (AIMs I-III). The specific experiments outlined in this proposal will exploit insect cell expression (i.e. Trichoplusia ni High Five cells), devoid of endogenous Na,K-ATPase, to address unresolved issues concerning the assembly, trafficking, and oligomeric state of the Na pump en route to the plasma membrane. Wild-type and mutant sheep alpha-subunits will be introduced into the insect cells via the baculovirus system and the pump maturation process will be followed from endoplasmic reticulum to plasma membrane by membrane fractionation and co-immunoprecipitation. The results from this work will provide important new information about the Na pump quaternary structure. The sodium pump is vital in a variety of organs for fluid and electrolyte balance. These processes are in dynamic equilibrium, and this equilibrium can become disrupted in a variety of disease states. In addition, it is becoming apparent that the Na pump contributes more to the cell than ionic homeostasis and the experiments proposed here are crucial if we hope to understand the complex cell biological role mediated by the Na pump.